The objective of the proposed work is to evaluate the role of oxidative stress in the development of human nuclear cataract, the most common type of lens opacity in older adults, and the type most likely to require surgery. The overall hypothesis of the proposal is that both molecular oxygen (O2) and UVA light can contribute to the formation of nuclear cataract. Aim 1 will investigate possible links between liquefaction of the vitreous humor (a common event occurring in the aging human eye), an increase in the level of O2 in the vitreous humor and nuclear cataract. This aim will employ enzyme-assisted liquefaction of vitreous humor in experimental animals, and the measurement of vitreal and lens 02 levels in vivo using a highly sensitive fiber optic device. Techniques will include slit-lamp biomicroscopy, laser scanning of lenses in vitro, a variety of biochemical analyses, SDS-PAGE, HPLC and electron microscopy to determine whether vitreous liquefaction can induce detrimental effects on the lens, leading to nuclear cataract. Two in vivo experimental animal models for nuclear cataract, hyperbaric O2 (HBO) and UVA light, will also be employed. Aim 2 will investigate the mechanism of O2-induced disulfide-crosslinking of lens crystallins, a modification strongly associated with human nuclear cataract. The Pi's hypothesis is that protein S-glutathiolation (the binding of glutathione to a protein) of lens crystallins protects against disulfide-crosslinking and crystallin insolubilization in an HBO/guinea pig in vivo model. Mass spectrometry and 2-D gel electrophoresis will be used to identify specific sites of glutathiolation, as well as specific crystallins that have become water- insoluble. The aim will be aided by the fact that sequences of all guinea pig lens crystallins are now available in the NEIBank on-line database. Aim 3 will test the hypothesis that UVA light in combination with O2 and a toxic UVA chromophore (such as that which can accumulate in the human lens nucleus with age) will generate H2O2, and induce aggregation of lens crystallins in vitro. This aim will employ guinea pig lens supernatants containing the endogenous UVA chromophore ^-crystallin with bound NADPH. Selected experiments will be conducted with other UVA chromophores including free NADPH, free kynurenine and a synthetic kynurenine peptide to mimic UVA chromophores present in the human lens. The aim will also determine whether GSH, as well as recombinant a-crystallin, can protect against UVA-induced aggregation of lens crystallins. Relevance: The studies are designed to elucidate the mechanism of formation of maturity-onset nuclear cataract, which is the cause for a major proportion of the 1.5 million cataract surgeries conducted in the United States each year. The results will provide valuable information on protecting the aging human lens against oxygen- and UVA-induced damage, and on guarding against formation of nuclear cataract.